Antiangiogenic tyrosine kinase inhibitors have differential efficacy in clear cell renal cell carcinoma in bone.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney neoplasm; bone metastasis (BM) develops in 35-40% of metastatic patients and results in substantial morbidity and mortality, as well as medical costs. A key feature of ccRCC is the loss of function of the von Hippel-Lindau (VHL) protein, which enhances angiogenesis via vascular endothelial growth factor release. Consequently, anti-angiogenic tyrosine kinase inhibitors (TKIs) emerged as a treatment for ccRCC. However, limited data about their efficacy in BM is available, and no systematic comparisons have been performed. We developed mouse models of bone and lung ccRCC tumors and compared their anti-cancer efficacy, impact on mouse survival, and mechanisms of action, including effects on tumor cells and both immune and non-immune (blood vessels, osteoclasts) bone stromal components. This approach elucidates the efficacy of TKIs in ccRCC bone tumors to support rational interrogation and development of therapies.

Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses.

Most platforms used for the molecular reconstruction of the tumor-immune microenvironment (TIME) of a solid tumor fail to explore the spatial context of the three-dimensional (3D) space of the tumor at a single-cell resolution, and thus lack information about cell-cell or cell-extracellular matrix (ECM) interactions. To address this issue, a pipeline which integrated multiplex spatially resolved multi-omics platforms was developed to identify crosstalk signaling networks among various cell types and the ECM in the 3D TIME of two FFPE (formalin-fixed paraffin embedded) gynecologic tumor samples. These platforms include non-targeted mass spectrometry imaging (glycans, metabolites, and peptides) and Stereo-seq (spatial transcriptomics) and targeted seqIF (IHC proteomics). The spatially resolved imaging data in a two- and three-dimensional space demonstrated various cellular neighborhoods in both samples. The collection of spatially resolved analytes in a voxel (3D pixel) across serial sections of the tissue was also demonstrated. Data collected from this analytical pipeline were used to construct spatial 3D maps with single-cell resolution, which revealed cell identity, activation, and energized status. These maps will provide not only insights into the molecular basis of spatial cell heterogeneity in the TIME, but also novel predictive biomarkers and therapeutic targets, which can improve patient survival rates.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) AML patients. Cells co-expressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated AML patients, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) patients with AML. Cells coexpressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T-cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated patients with AML, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.